The present invention, in some embodiments thereof, relates to methods and, kits for isolating membrane proteins.
Isolation of membrane proteins (MPs) in a pure, concentrated and functional state is a precondition for structural determination by X-ray crystallography, electron microscopy or nuclear magnetic resonance (NMR).
Extraction of membrane proteins from the membrane of a cell in which they are expressed may be achieved by addition of detergents at concentrations greater than their critical micellar concentration (cmc). Under these conditions, the detergent disrupts the membrane and, in parallel, surrounds and covers the hydrophobic domains of the protein, leading to formation of water-soluble [detergent-MP-lipid] ternary complexes. Purification is accomplished either via classical chromatographic methods (e.g. ion exchange chromatography) or by genetically engineered affinity-tags (e.g. His-tag) which can lead to highly pure protein preparations. Clearly, exclusion of other (non-membrane) cellular proteins by non-chromatographic means prior to the chromatographic step, would simplify purification and potentially lead to higher recovery yields and overall greater purity.
In 1981, Bordier demonstrated that MPs (being hydrophobic) partition efficiently into detergent-rich phases composed of the non-ionic detergent Triton X-114, whereas water-soluble proteins do not. This partitioning process, called cloud point extraction, relied on the ability of Triton X-114 to undergo phase separation at ˜22° C. into detergent-rich and detergent-poor phases. Although Triton X-114 provided working conditions that could preserve the functionality of many MPs, the approach was limited by the fact that numerous other detergents, commonly used in the purification of membrane proteins, only reach the cloud point at elevated temperatures that would denature most proteins. Successful attempts to lower the cloud point temperature in the presence of high concentrations of water-soluble polymers have been reported. However, it is clear that purification would be greatly simplified if neither polymers nor precipitants were required.
Background art includes Patchornik et al., Journal of Colloid and Interface Science 388 (2012) 300-305; and Patchornik et al., Soft Matter, 2012, 8, 8456.
Additional background art includes WO2005/010141, WO2006/085321 and WO2009/010976.